longitudinal dynamics of body weight change in the development of type 2 diabetes

Longitudinal Dynamics of Body WeightChangeintheDevelopmentofType2DiabetesBarbara C. Hansen1,2,3, Jennifer D. Newcomb1,2, Ren Chen4 and Ellen H. Linden1,2

Objective: The aim of this study was to test the obesity–type 2 diabetes mellitus (T2DM) link in the

context of longitudinal changes in body weight during the progression to diabetes in mature adult

nonhuman primates (NHP).

Design and Methods: A colony of 245 adult rhesus monkeys aged 8-41 years with 179 males were

used to define overweight in males as a body weight: �13.5 kg or body fat (BF) �18% and

obesity as �16.5 kg or BF �27%, and overweight in nonpregnant females was identified as a

body weight >8.5 kg or BF >21% and obesity as �10.5 kg or BF �30%. A subgroup of 48 males

(24 T2DM and 24 age-matched non-T2DM) males were studied before and following the onset of

overt T2DM for the effects of changes in body weight and obesity in inducing this conversion to

overt T2DM.

Results: Three years before overt T2DM, mean body weight was 18.4 6 3.3 kg. The DM-destined group

body weight was 3.2 6 1.1 kg greater and had a longer duration and greater severity of obesity, with

peak body weight reached at 3.2 6 1.8 years before overt T2DM. At DM onset the two groups did not

differ significantly in body weight or adiposity.

Conclusions: The natural progression from pre-DM to overt T2DM is caused neither by the amount of

excess body weight at DM onset nor by the proximate increases in body weight/adiposity during the pre-

DM period of impaired glucose tolerance. Obesity was, however, essential preceding all NHP cases that

developed T2DM.

Obesity (2013) 21, 1643-1649. doi:10.1002/oby.20292

IntroductionIncreased adiposity is considered one of the major risk factors that con-

tributes to or facilitates the development of the metabolic syndrome, type

2 diabetes mellitus (T2DM), cardiovascular disease, stroke, and cancer

(1-24). A meta-analysis of obesity and its comorbidities by Guh et al. an-

alyzed 89 articles and identified 18 different overweight and obesity-

associated comorbidities, among them, T2DM, endometrial and prostate

cancer, coronary heart disease, and stroke in both males and females (6).

The Guh analyses, using BMI as the primary variable, showed that

T2DM was strongly associated with overweight in males (relative risk

[RR] ¼ 2.40 (2.12-2.72)) and in females (RR ¼ 3.92 (3.10-4.97)), and

even more strongly with obesity in males (RR¼ 6.74 (5.55-8.19)) and in

females (RR ¼ 12.41 (9.03-17.06)) (6). A large cohort study of 121,700

middle-aged (33-55 years) females showed that body weight, identified

by increased BMI, is the single most important contributing factor for the

development of T2DM (7). The relationship between gain in weight or

weight change and the development of diabetes was not addressed.

NHPs frequently develop spontaneous obesity, metabolic syndrome,

and T2DM (8-27) with characteristics that are extraordinarily like

those of humans (25). The present colony of rhesus monkeys has

been studied longitudinally for many years under constant dietary,

activity, and environmental conditions. This consistency is a key

consideration in discerning the characteristics of the DM progression

and factors contributing to its overt expression. Therefore, these

monkeys serve as an excellent animal model for longitudinal analy-

sis of the pathophysiology and natural trajectories of obesity, meta-

bolic syndrome, and T2DM.

The aims of this study were to further characterize the increased adi-

posity and obesity of middle-aged male and female macaques and

the relationships between changes in adiposity and the development

of T2DM under constant dietary and environmental conditions.

Interactions between changes in body weight/adiposity and the de-

velopment of T2DM were examined in a subgroup of males that

1 Obesity, Diabetes and Aging Research Center and the Center for Preclinical Research, College of Medicine, University of South Florida, Tampa, Florida, USA.Correspondence: Barbara C. Hansen ([email protected]) 2 Department of Internal Medicine, College of Medicine, University of South Florida, Tampa, Florida,USA 3 Department of Pediatrics, College of Medicine, University of South Florida, Tampa, Florida, USA 4 Office of Research, College of Medicine, Universityof South Florida, Tampa, Florida, USA

Funding agencies: This work was supported by NIA N01AG31012 and NIA HHSN2532008002C.

Disclosure The authors declared no conflict of interest.

Received: 22 July 2011 Accepted: 26 November 2011 Published online 2 January 2013. doi:10.1002/oby.20292

www.obesityjournal.org Obesity | VOLUME 21 | NUMBER 8 | AUGUST 2013 1643

Original ArticleOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Obesity

developed diabetes while under longitudinal study and compared to age-

matched nondiabetic adult rhesus monkeys that did not develop T2DM,

which is very difficult to do in free living human subjects.

MethodsPrimate colony characteristics and careThis rhesus monkey colony (Macaca mulatta) consisted of 245 indi-

vidually housed monkeys (73% males for historical reasons). Mon-

keys were housed according to the Guide for the Care and Use of

Laboratory Animals (14). All primate care and experimental proto-

cols were reviewed and approved by the Institutional Animal Care

and Use Committee. The monkeys were provided with extensive

environmental enrichment, such as treats, toys, music, videos, and

human contact. The environmental conditions were consistent for all

monkeys, and food intake was determined daily. All monkeys were

fed ad libitum on a primate diet (Lab Diet 5038, Richmond, IN)

(13.1% calories from fat, 18.2% calories from protein, and 68.7%

calories from carbohydrates) and had access to water at all times.

The males ranged in age from 8.8 to 34.6 years and the females

ranged from 8.8 to 41 years. Male body weights ranged from 8.8 to

26.5 kg, plus two outlier males weighing �31 kg. At a mean age of

16 years, the average male body weight was 14 kg. Among the males

that had not lost weight because of aging or diabetes, the mean body

weight was 17 kg. Among the females body weights ranged from 5.7

to 13.6 kg and at a mean age of 20 years, the mean body weight was

8.2 kg. Because of the major sexual dimorphism, males and females

could not be combined in weight or body composition analyses. The

colony has included 113 DM monkeys, with an average onset of DM

at 18.8 6 4.8 years (range of age of onset: 9.8-30.5 years), and the

oldest insulin-requiring DM monkey was aged 41 years.

Dual-energy X-ray absorptiometry analysisImaging by dual-energy X-ray absorptiometry (DXA) scan (Lunar

Prodigy Pro, Piscataway, NJ) was performed to allow accurate deter-

mination of total body fat (FM) and the total body fat free mass

(FFM) with precise determination of changes in body composition

across time. Two hundred nine DXA scans were performed on 97

rhesus monkeys (males n ¼ 76, females n ¼ 21; age range 8-30

years). The numbers available in the male group enabled the statisti-

cal analysis of the % BF and FFM compared to body weight across

three subgroups of male monkeys: (1) normal monkeys (non-DM)

(apparently normal with no evidence of metabolic disturbances and

no diabetes); (2) prediabetic monkeys (pre-DM) (with all in this

group meeting two or more of the following three criteria: weight

� 13.6 kg in males, Kgluc � 2, and/or triglycerides � 150 mg/day);

and (3) diabetic (DM) monkeys (defined as having two or more fast-

ing plasma glucose (FPG) levels �126 mg/dL).

Longitudinal prospective data analysis of bodyweight changes in relation to the progression toovert diabetesOn the basis of available longitudinal data obtained before overt dia-

betes, 24 male rhesus monkeys were selected from the 113 T2DM

population, and these were compared to 24 age-matched males who

never became diabetic and had been studied in the laboratory over

the same age range (total of 48 monkeys).

ProceduresAfter an overnight 16-hour fast all blood samples were collected via

peripheral intravenous catheters under anesthesia with ketamine (10

mg/kg body weight). Plasma glucose concentration was determined

by the glucose oxidase method (glucose auto-analyzer, Beckman

Instruments, Fullerton, CA and Analox Instruments, London, UK).

Statistics and analysisAll results are presented as means 6 SD. Linear regression was

used to determine the presence of a significant change in body

weight, % BF, and/or FFM in non-DM, pre-DM, or DM rhesus

monkeys. The Pearson correlation coefficient was used to test the

relationships between body weight and age in relation to FFM and

% BF in the 97 monkeys having body composition analyses. A lin-

ear mixed effect model and paired t-tests were used to determine

body weight changes in the DM and non-DM age-matched groups at

each 1-year period. Statistical analysis was performed using SPSS,

17.0 software. A P value of �0.05 was considered statistically

significant.

ResultsBody weight and body composition analysisusing DXA scansOn the basis of extensive longitudinal data in this colony of adult

rhesus monkeys, we have identified the following criteria for classi-

fying fully adult lean, overweight, and obese female rhesus: lean

(body weight < 8.5 kg or % BF < 21), overweight (�8.5 kg or %

BF � 21), and obese (� 10.5 kg or % BF � 30). Male rhesus mon-

keys were considered lean if the body weight was <13.5 kg. This

weight was associated with % BF < 18%. Overweight was defined

as �13.5 kg, a weight range associated with a BF of about 18.1-

26.9%. Obese male rhesus had body weights �16.6 kg. BF was usu-

ally >27% in monkeys above this weight (Table 1).

In adult male monkeys over the age of 8.8 years, body weight was

strongly positively correlated with % BF (r ¼ 0.80, P < 0.001) and

with FFM (r ¼ 0.75, P < 0.001) (Figure 1A and B). Among the

males there was also a positive association between FFM and % BF

(r ¼ 0.31, P < 0.001) (Figure 1C). Among the small group of

female monkeys over the age of 8.2 years (N ¼ 21), body weight

TABLE 1 Weight category of lean, overweight, and obesenonhuman primates (N 5 113)

Weight

category

Female Male

Body

weight (kg)

Body

fat (%)1Body

weight (kg)

Body

fat (%)

Lean <8.5 <21 <13.5 <18

Overweight �8.5 �21 �13.5 �18

Obese �10.5 �30 �16.5 �27

1Percent fat represents the usual amount of fat at the given weight by sex, but indi-vidual variability means there may be some deviations from the estimated fat basedon weight and vice versa.

Obesity Body Weight Change in the Development of T2DM Hansen et al.

1644 Obesity | VOLUME 21 | NUMBER 8 | AUGUST 2013 www.obesityjournal.org

was positively correlated both with % BF (r ¼ 0.85, P < 0.001) and

with FFM (r ¼ 0.73, P < 0.001).

When adult males were classified by metabolic status as nondiabetic

(non-DM), pre-diabetic (pre-DM), or diabetic (DM), adult male

monkey weights ranged widely within group as shown in Figure 2:

Non-DM mean (6SD) body weight was 14.4 6 3.1 kg, % BF

23.2 6 11.4, and FFM 9.9 6 1.6 kg. Pre-DM body weight was

17.2 6 2.8 kg with an average % BF of 33.0 6 6.7 and FFM of

11.3 6 1.5 kg. DM body weight was 16.7 6 2.8 kg, % BF 32.6 6

6.7, and FFM 10.9 6 1.4 kg. There were no differences between the

body compositions of the pre-DM and DM groups; however, both

groups were significantly heavier and fatter than the non-DM group.

Because the T2DM is a disease associated with aging, we examined

the effect of age on weight, % BF, and FFM in adult male monkeys

(Figure 2A-C). In males aged 8-30 years, there was no association

between age and body weight or age and FFM (Figure 2A and C).

Because of the tendency of younger adult monkeys (8-15 years) to

increase in % BF and older monkeys (15-40 years) to decrease in %

FIGURE 1 Body weight, % body fat, and lean body mass in adult male rhesus monkeys over the age of 8years, including normal animals (^). prediabetic (n), and overtly diabetic monkeys (~). Body weight was sig-nificantly and positively correlated with % BF (r ¼ 0.80, P < 0.001) (A) and with FFM (r ¼ 0.75, P < 0.001(B). FFM and % BF were also associated (r ¼ 0.31, P < 0.001) (C).

Original Article ObesityOBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY


BF there is a parabolic relationship between age and % BF in the

male NHP population (Figure 2B).

Among the female monkeys (data not shown), the DM monkeys

were significantly lower in body weight (P < 0.05) and % BF (P <0.01) compared to pre-DM monkeys. The relatively low BF of the

overtly diabetic female animals reflected a much longer period of

gradual weight loss after the development of diabetes and was asso-

ciated with the severity of diabetes. One female with >10 years of

insulin-requiring DM lived to be 40 years of age. Among the female

monkeys (data not shown), the DM monkeys were significantly

lower in body weight (P < 0.05) and % BF (P < 0.01) compared to

pre-DM monkeys. The relatively low BF of the overtly diabetic

female animals reflected a much longer period of gradual weight

loss after the development of diabetes and was associated with the

severity of diabetes.

The relationship between body weight and theonset of DM (FPG � 126)The mean body weight of the 24 longitudinally studied male DM

monkeys, 5 years before meeting the diagnostic criteria for overt

FIGURE 2 In fully adult male monkeys over the age of 8 years, there was no association between age andweight (A), nor between age and % body fat, and between age and FFM (B). There was parabolic associ-ation between % BF and age (when the age range 3-30 years was considered; dashed line) and a nega-tive association among the pre-DM and DM (22-30 years).



T2DM, was 17.3 6 3.4 kg. Of these monkeys, 11 monkeys achieved

their own peak body weights 3 years (18.6 6 3.3 years) before

reaching the diagnosis of overt T2DM. These peak body weights

were reached at a wide range of ages (12.6-21.9 years). The individ-

ual weight trajectories of the eight male monkeys that were studied

for at least 5 years before T2DM diagnosis are shown in Figure 3A.

All monkeys lost weight before reaching overt T2DM and during

the first year post-DM diagnosis, with a mean weight loss during

FIGURE 3 Longitudinal analysis of the change in mean body weight of eight DM monkeys from 5 yearsbefore the diagnosis of DM (FPG � 126 mg/dL) to 1 year after DM diagnosis (no insulin treatment) (A)compared to the mean change in body weight of eight age-matched non-DM monkeys (B). The meanbody weight of 24 DM monkeys and 24 age-matched non-DM monkeys were represented as mean 6

SD (C). There was a significant decrease in body weight during time �1 to 0 (P < 0.0001) and duringtime 0-1 (P ¼ 0.0009) in DM group compared with the age-matched non-DM group. Only one failed soshow weight loss during the 5-year period preceding overt T2DM.



this first DM year of �1.4 6 1.1 kg, preceding initiation of insulin

treatment (Figure 3A). The mean body weight at the end of the

study was 14.3 6 0.7 kg with an average �3.2 kg weight loss over

the progression from pre-DM to overt DM and for 1 year beyond.

No male rhesus monkey developed T2DM without having reached a

body weight of at least 14.1 kg and a BF content of >22%. There-

fore, to date no longitudinally studied rhesus (male or female) has

been observed to develop T2DM unless the monkey had been at

least overweight (or obese). No rhesus had been identified under adlibitum fed conditions to be lean at the time of onset T2DM rhesus;

thus, overweight or obesity preceded all cases of T2DM among rhe-

sus monkeys. Weight loss, however, clearly occurred during the late

progression to diabetes, and in some cases was very significant

before meeting the threshold for the diagnosis of T2DM.

The 24 age-matched non-DM monkeys, studied over the same time

period as those that developed T2DM while under longitudinal

study, had a mean body weight of 15 6 1.4 kg at the beginning of

the study period and a mean of 16.5 6 1.1 kg at the end of the pe-

riod. The non-DM monkeys, age-matched to the DM monkeys,

tended to gain less weight compared to the DM monkeys; however,

the weight gain varied between monkeys, as shown in Figure 3B,

for the eight non-DM monkeys age matched to the DM monkeys in

Figure 3A. The average body weight trajectories for the DM and the

non-DM monkeys beginning 5 years before DM and extending to 1

year after DM diagnosis are shown in Figure 3C. Statistical analysis

showed no significant difference in weight change between the DM

group and the age-matched non-DM group during the time period

�5 to �1 years, P ¼ 0.6361 because this was on average a steady-

state period; some were gaining; some losing; and some were stable.

From 1 year before DM onset until overt T2DM, there was a signifi-

cant weight loss in the DM group compared with the age-matched

non-DM group, P < 0.0001. In addition, the DM group showed a

further significant decrease in body weight over the year following

DM onset compared to the age-matched non-DM group, P ¼ 0.0009

(Figure 3C).

DiscussionTo develop new standards for overweight and obesity in rhesus

NHPs, and to use these in examining the present colony (and for

future prospective use in larger colonies without DXA analyses

available), we have measured body weight, % BF, and FFM. Meas-

urements were performed using DXA scans in both male (age 8-30

years) and female (age 8-30 years), non-DM, pre-DM, and DM rhe-

sus monkeys. Before overt DM, in both males and females, the non-

DM monkeys were significantly lower in body weight (P < 0.01),

% BF (P < 0.05), and FFM (P < 0.05) compared to the pre-DM

monkeys.

The average age of onset of T2DM in this colony was 18.8 6 4.8

years (range 9.8-30.5 years). Nonhuman primates provide the unique

possibility of examining directly and quantitatively the longitudinal

changes in body weight and obesity combined with the timing of

the onset of T2DM. Various studies have shown the greater accu-

racy and reliability of using the DXA scan in estimating BF and

FFM when compared with other methods of estimating BF and

FFM, such as the titrated water method (isotope dilution of tritium

[3H2O]), total body potassium, bioelectrical impedance analysis, and

skin fold measurements both in humans and rhesus monkeys (10,15-

19). In this study, the relationship between body weight and body

composition in males and females, and in non-DM, pre-DM, and

DM male rhesus monkeys has been assessed using DXA scans (Fig-

ure 1). We have shown that in non-DM monkeys, % BF in both

males and females and FFM only in males were significantly lower

compared to pre-DM monkeys. Body weight was also significantly

associated with both % BF and FFM in male and female monkeys.

In addition, the body FFM significantly decreased with age when all

monkeys were combined; however, % BF decreased with age princi-

pally in monkeys in the age range from 22 to 33 years (or older)

(data not shown). Our current results confirm our previous report

that showed significant associations between body weight and % BF

in a small group of rhesus monkeys measured by the titrated water

dilution method (N ¼ 24, obese/nonobese monkeys) (10). This study

also agrees with Black et al., who studied relatively lean (6.7-12.3

kg) and young (5-8 years) male rhesus monkeys (N ¼ 10) showing

significant correlations between total body weight, fat mass, and

lean mass in rhesus monkeys using DXA measurements (15).

In this study, body weight changes were analyzed over 1-5 years

before the development of T2DM in rhesus male monkeys and com-

pared to the body weight changes in age-matched male monkeys

that never developed T2DM. Monkeys that developed T2DM had

shown considerable weight loss over several years before the devel-

opment of T2DM and well in advance of the increase in FPG to

overt T2DM levels (>126 mg/dL). Monnier et al.’s study on early

signs of diabetes using 24-hour glucose monitoring in 130 T2DM

patients with variable DM severity concluded that the first sign of

diabetes in humans with T2DM was postprandial glycemia (20,21),

while the fasting glucose was still below the diagnostic level of

overt T2DM (6.3 mmol, 114 mg/dL) (20,21). They thus considered

fasting hyperglycemia to be a late indicator (immediately preceding

overt DM) of T2DM and prandial induced hyperglycemia leading

by several years in the progression to T2DM. The weight loss

observed in this study was most likely due to the intermittent post-

prandial hyperglycemia that developed �1-3 years before the

appearance of fasting hyperglycemia. In humans and monkeys, such

prediagnosis postprandial hyperglycemia has been reported fre-

quently and also associated with increased glycosuria.

At the beginning of this study, monkeys that developed T2DM were

significantly overweight (3.2 years before overt T2DM) compared to

age-matched monkeys that never developed T2DM. Both modest

(<10 kg) and substantial (>10 kg) weight gain by 8.8 years of age

was associated with increased risk of the development of overt

T2DM in subjects with normal glucose levels (<6.1 mmol, �110

mg/dL) (22). Neither moderate nor substantial weight gain increased

the risk of T2DM in human or monkey subjects with high glucose

(>6.1 mmol, >110 mg/dL) (22) probably because, as shown in this

longitudinal study of rhesus, they were more advanced in their dis-

ease and some were losing weight.

In addition, duration of obesity increased the risk for development

of DM in humans (23). There was a fourfold increase in the relative

risk of the development of T2DM in human subjects who showed

severe obesity (BMI � 30) for less than 5 years. However, human

subjects with severe obesity with duration lasting over 5 years

showed an eightfold increase in relative risk of development of

T2DM (22). Therefore, baseline glucose level, degree of obesity,

and duration of obesity increased risk for development of T2DM in

humans. Interestingly, in this study, non-DM monkeys that



continued to gain weight (peak body weight greater than their

T2DM-matched peak body weight) over the 3 year beyond the study

period eventually developed T2DM. In addition, all pre-DM mon-

keys were classified as obese; thus, as in humans, increased weight

or excess adiposity is not a sufficient early predictor of overt

T2DM.

ConclusionChange in body weight/BF was not the precipitating factor in deter-

mining progression to overt T2DM. However, increased adiposity

and body weight was present several years before overt DM in all

rhesus monkeys that eventually progressed to DM.

Weight loss was shown to be a preceding diagnostic sign of progres-

sion of overweight primates toward T2DM. The maximum body

weight occurred in most monkeys significantly before the onset of

diabetes, most commonly 3-5 years before the overt disease.O

VC 2013 The Obesity Society

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